This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. After determining the optimal dose of the third-generation live attenuated Listeria monocytogenes vector encoding SIV gag, termed Lmdd-BdopSIVgag, we examined a different dose-schedule, administering the vaccine (3 x1012 colony-forming units (CFU)) intragastrically every other day for 4 doses at weeks 0 and 12 to five rhesus monkeys. Controls received the "empty" vector, Lmdd-Bdop. However, the analysis showed that this every-other-day schedule was not as good as administering the vaccine daily for three consecutive days. To test whether the vaccinated animals had been tolerized by the repeated exposure to the Listeria vaccine, we re-immunized them with the same Lmdd-BdopSIVgag according to the prior daily x 3 schedule;this group of monkeys was designated Group 1B. A new group of 5 na[unreadable]ve monkeys received the vaccine in parallel (Group 1A). New na[unreadable]ve controls (Group 2;5 monkeys) received only empty vector. Good cellular immunity was seen in Groups 1A as well as 1B, effectively ruling out tolerization. Both Groups then received two mucosal boosts with live attuenated adenovirus encoding SIV Gag (Ad5hrSIVGag), which resulted in strong increases in Gag-specific cellular immunity. Next, Groups 1a and 1B received protein immunizations consisting of trimeric HIV-1 gp160 and Tat with the aim to induce neutralizing antibody responses. At the conclusion of the protein boosts, the monkeys were challenged with 5 low, weekly intrarectal doses of a neutralization-sensitive R5 SHIV encoding a heterologous HIV clade C envelope. Substantial protection was seen, including prevention of systemic infection. We conclude that inducing balanced immune responses consisting of cellular as well as humoral immunity can be successful. Strong cellular immunity was induced with oral Lmdd-BdopSIVgag priming, followed by mucosal boosting with Ad5hrSIVGag, whereas cross-neutralzing antibodies were generated with HIV-1 gp160 and Tat. Our multi-modality vaccine approach that targeted several SHIV gene products is promising, even against heterologus virus challenge.